Cannabis Testing: An Industry Guide

Introduction:

Historically, the vast majority of countries have opted for the blanket prohibition of marijuana. But, after decades of stagnant legislation, this has begun to change at pace in recent years as several countries and US states have adopted, or are poised to adopt, more liberal approaches to regulation of the drug. These have ranged from the experimental – as seen with the nationwide legalization of cannabis in Uruguay – to the tentative – as in Canada which introduced laws permitting strictly regulated medicinal use over 15 years ago. Currently over half of all US states permit medicinal use of the drug and eight, as well as Washington DC, have opted to allow some recreational use for adults under state law.

These legislative changes have been driven by a marked shift in public attitudes towards the legal status of cannabis. For example, in the US, Gallup polls have shown an increase in support for marijuana legalization from 25% in 1996 to 60% in 2016. A 2016 survey of 1000 Canadians found that 70% were either supportive or somewhat supportive of marijuana legalization. And the figures are even higher when voters are asked about medicinal cannabis use specifically.

But moving away from the strict prohibition of cannabis bound in the laws of most countries is far from straightforward. When legislators shift away from this model, there are multiple alternatives to consider and there is no simple black and white, legal or illegal, stance available. Regulators must decide whether to opt for legalization or decriminalization, whether the laws apply to all forms of marijuana (for example dry marijuana or oils and extracts), what quantities and age limits are restricted, and how the laws apply separately to the growth, possession and sales of the drug. Even countries like Uruguay, which have opted for widespread legalization, must decide on a commercial model for legal sales of the drug.

This year’s Pittcon, taking place in Chicago from 5-9 March, 2017, will be host to a number of symposia that will get to grips with the issues emerging from the rapidly developing cannabis industry from a medical, legal, social and analytical point-of-view. This includes talks covering the current state of research into cannabis and derivatives for medical purposes, a look at the social stigma of cannabis and whether this is holding back medical research, as well as a wealth of advances in analytical methods for characterizing cannabis, which will play a role in quality control, regulation and pharmaceutical research.

For example, the session “It’s Legal! Now What?” will look at the challenges facing laboratories in the era of legalized cannabis. What approaches are available for analyzing cannabis samples? How can we stay ahead in the detection of contaminants and adulterants? How can we develop and enforce laboratory standards to ensure medicinal cannabis is safe for patients? This symposium will feature speakers from the Colorado Department of Public Health and Department of Agriculture, one of the few US states to allow recreational cannabis use, and the Association of Commercial Cannabis Laboratories.

And for an update on the use of cannabis for medical purposes, Tracy Ryan from Cannakids, a company that provides cannabinoid-based treatments for children with serious illness, will be speaking during the symposium “Analytical Cannabis II”. Ryan will present anecdotal and clinical trial results of medical cannabis for children and adults, and discuss the range of conditions, such as epilepsy, Crohn’s disease and PTSD, that could stand to benefit from cannabis treatment.

The US Food & Drug Administration has stated that it supports research into the medical use of marijuana, although it has not approved the drug for any indication. During this symposium, Uma Dhanabalan from TotalHealthCareTHC, will ask whether the continued stigma surrounding cannabis is holding back research in this field, and argues that greater awareness and fewer misconceptions will help realize the full potential of cannabis as a medicine.

In the chapters to come, we will be taking a look at some of these topics in greater detail, and what highlights to expect from this year’s Pittcon. This includes analytical methods for characterizing active ingredients and contaminants in cannabis, the development of industry laboratory standards, methods of cannabis extraction, as well as cannabis detection for law enforcement purposes. With major industry players, including Sigma Aldrich, Shimadzu, PerkinElmer, Restek and CEM Analytical, already confirmed for this year’s exhibition, Pittcon 2017 will be a one-stop destination to hear about all the latest developments in cannabis analysis.

Chapter 1 – Active Ingredients in Cannabis

Tetrahydrocannabinol (THC) is the major psychoactive ingredient in cannabis and has consequently received much attention from scientists. However, it has emerged from research that cannabis actually consists in excess of 500 chemical entities and around 60 of these come from the same family as THC – the cannabinoids. Another important family of compounds present in cannabis are terpenes and terpenoids which give cannabis its distinctive flavor and aroma. Both terpenes and other cannabinoids can interact with THC to enhance or antagonize its psychoactive effects.

Potency testing

Verifying the potency of cannabis is key for cannabis testing labs, and can be achieved through LC and GC methods. Restek’s Raptor LC columns can be used alongside any HPLC instrument, accelerating the analysis time without the need for UHPLC equipment. The company says that their high-throughput approach can complete cannabinoid analysis in 3.7 minutes. They have also shown that their Rxi®-35Sil MS column can be used alongside GC equipment to rapidly analyze cannabinoids in a matter of minutes.

Another approach that has the potential to offer cannabis producers access to on-the-spot potency testing is Fourier transform mid-infrared (FT-IR) spectroscopy. A team from PerkinElmer tested the method on intact and ground cannabis bud samples. They showed that the method could accurately quantify the levels of tetrahydrocannabinolic acid which is converted to THC upon heating and, another important cannabinoid, cannabidiolic acid (CBDA) which is converted to cannabidiol. The researchers also demonstrated that FT-IR could detect changes in cannabinoid concentrations according to growing time and conditions, showing that it has the potential to allow cannabis producers to monitor and optimize conditions and determining harvest time.

Steep Hill, also presenting at Pittcon 2017, have developed a cannabis analyzer called the QuantaCann2 that uses near-infrared (NIR) spectroscopy. The company argue that the method, although less versatile, has advantages over other spectral approaches that are destructive, require more sample preparation, greater operator expertise, and the use of solvents. The team that developed the device have shown that it can quantify four major cannabinoids in cannabis – CBDA, cannabidiol, THCA and THC to within 0.7%, 0.4%, 1.3% and 0.6% accuracy, respectively, when compared with HPLC reference spectra.

Terpene profiling

Cannabis contains a complex profile of terpenes which are thought to be responsible for some of the drug’s purported health benefits. As this profile varies between strains, from crop to crop and even plant to plant, it can also be used for quality control purposes. An increased interest in characterizing terpene profiles has followed from the wider legalization of cannabis and is required in some states by law.

Several companies have developed approaches to terpene profiling. For example, Shimadzu, who will be at this year’s Pittcon, created a method using their mass spectrometer with a full evaporation headspace technique (FET) to overcome the fact that plant material does not dissolve in solvent. The Shimadzu team have shown that using a single-phase liquid-gas system they could quantify the presence of terpenes in accordance with Nevada state law in three different strains of cannabis.

Restek have also generated a workflow for separating terpenes using a headspace gas chromatography-flame ionization detection (GC-FID). Like Shimadzu’s approach, the method also uses FET with a single-phase gas system. They have demonstrated the efficacy of the method in characterizing the terpene profile of pelletized hops, as a proxy for cannabis. They used the Shimadzu Rxi®-624Sil MS column, which has a small-bore configuration, and can also be used for analyzing residual solvents in cannabis, using the same setup and technique.

Cannabis component analysis at Pittcon 2017

At Pittcon 2017, Scott Kuzdzal from Shimadzu Scientific Instruments will introduce the symposium ‘Analytical Cannabis II’. In the session ‘Current and Future Analytical Technologies for Cannabis Testing and Research’, Kuzdzal will discuss the many different chemical compounds found in cannabis and some of their reported health benefits. He will also outline why cannabis testing labs are so important for quality control in the era of medical cannabis, including the measurement of terpene concentrations, and the detection of contaminants such as pesticides, heavy metals and mycotoxins. Kuzdzal will also consider how analytical technologies are enhancing quality control testing including in clinical settings.

In an oral session, the conference will also hear from Laura McGregor of Markes International who will describe how two-dimensional gas chromatography couple with time-of-flight mass spectroscopy can aid the analysis of complex plant-based samples, such as cannabis. McGregor will also outline how the approach of tandem ionization could help researchers to keep pace with the emergence of so-called legal highs by boosting researchers’ ability to identify novel structures for which no reference spectra are available.

Also in attendance at this year’s conference will be Sigma-Aldrich who offer a range of solutions for cannabis testing labs. This includes the company’s Ascentis Express columns which can be coupled to any HPLC, UHPLC or LC-MS instrument allowing the characterization of a sample’s active ingredients, including cannabinoids.

Advion will also be at Pittcon 2017 to present their compact mass spectrometer. The device, which is much smaller and priced lower than a standard MS system is designed to increase the accessibility of MS to labs, particularly those with restricted space. Advion have shown that compact MS can be applied to the analysis of cannabinoids alongside thin layer chromatography for qualitative detection of cannabinoids and alongside HPLC for quantitative determination.

With the market for cannabis growing along with expanding legalization, there is increasing demand for cannabis extract products. This includes oils and waxes that can be used in vaping, drinks, and edibles. For both recreational cannabis users seeking a high and for potential medicinal applications, there is also growing interest in purified forms of cannabis such as cannabinoid isolates.

Methods of extraction

Traditionally butane extraction has been the most commonly used method of cannabis extraction, but it is being used less and less in commercial settings these days due to the risk of explosions from the highly flammable gas. However, the technique has apparently been growing in popularity among illegal cannabis producers who use it to create high-strength butane hash oil (BHO). In the UK, two people have died and 27 people have been injured in the process of generating BHO over the last two years, according to police reports.

In lab settings, the most popular approach is solvent extraction, which requires further purification steps to remove any residual solvents.

Another method, which is more expensive but gaining more widespread use, is supercritical fluids chromatography or CO2 extraction, which uses carbon dioxide as a solvent and therefore doesn’t require as much post-processing.

At this year’s Pittcon, Xiaoning Lu from Sigma-Aldrich will present another technique that could be applied to cannabis extraction – online solid phase extraction (SPE). This method is widely used by labs to isolate analytes from complex matrices but off-line methods are time consuming, labor-intensive and have poor reproducibility.

Lu will present research carried out on thyroid hormone samples in biological matrices using an online-SPE cartridge developed by the company, alongside liquid chromatography/mass spectrometry (LC/MS). He will show how the online cartridge was able to significantly boost the LC/MS response and improve reproducibility, and will also explain how the same cartridge can be applied in the detection of cannabis analytes in blood serum and urine samples.

Removing residual solvents

When solvent-based extraction processes are used, the cannabis extract must undergo further steps to remove any residual solvents, as these can be harmful to human health. It is therefore vital to verify that the solvents have been completely removed and there are a number of chromatographic options available for doing so.

Commonly used is gas chromatography (GC) and static headspace GC can be used to concentrate volatile analytes for analysis and provide rapid identification and quantification of residual solvents. Shimadzu and Sigma-Aldrich, who will both be presenting at this year’s Pittcon 2017 offer such solutions for residual solvent analysis.

Also at Pittcon 2017, Robert Driscoll from Robatel Inc., a Massachusetts-based centrifuge provider, will discuss centrifugal chromatography as an approach to isolating components from organic samples. Driscoll will outline the benefits of fast centrifugal partitioning chromatography – a method that allows components with similar molecular structures to be isolated from a sample – over other techniques available. He will also discuss recent advances in the design of the technology and its use in isolation of cannabis as well as tobacco, opiate derivatives and nutraceuticals.

Chapter 3 – Cannabis Testing – Identifying Chemicals and Contaminants

In the United States, although recreational or medical use has been authorized by several states, cannabis is still illegal at the federal level. Consequently, the Food & Drug Administration (FDA) does not recognize cannabis as a regulated product and does not provide any standards for its cultivation and supply. This raises the risk that cannabis products could be contaminated, potentially endangering consumers’ health.

This is particularly pertinent for those accessing medical cannabis who may have compromised immune systems. Fortunately, there are a number of analytical techniques that have been applied in clinical, pharmaceutical, food safety and environmental settings that are equally effective for verifying the absence of chemicals and contaminants in cannabis samples.

At Pittcon 2017, Joshua Crossney from jCanna, Inc., a non-profit organization interested in improving cannabis analytical testing technologies, will discuss how the emerging cannabis testing and research industries are benefiting from collaborating and sharing the knowledge and experience of these other more long-standing industries.

At this year’s conference, you can also hear about some of the specific techniques and the latest methods being developed to assist the provision of safe, uncontaminated products in the new era of legalized cannabis.

Pesticides

Cannabis plants are susceptible to a number of bacteria, fungi, yeasts and molds but many pesticides available to try to prevent infestation can themselves be harmful to human health. And while pesticide use on other crops is federally regulated, this is not the case for cannabis. What’s more, no pesticides have even been tested or registered for use on cannabis. As a result, individual states that have legalized cannabis are having to come up with their own regulations for pesticide use and eliminating contamination from the final product.

This year’s Pittcon will feature discussion of a number of techniques now available to help meet the challenge of cannabis quality control. Attending the conference will be Restek, Shimadzu and Sigma Aldrich, who all provide LC-MS and GC-MS solutions for pesticide analysis. There will also be a presentation from Julie Kowalski of Restek who will outline a modified QuEChERS technique – which is already popular for pesticide testing in food and agriculture – using LC-MS/MS to analyze pesticide residues that has so far been applied to over 200 pesticide types.

Jack Henion from Advion will also discuss how mass spectrometry using the company’s compact device could be used as a screening method to test the quality and purity of cannabinoid products and detect the presence of pesticides and other contaminants.

Other analytes and contaminants

Another trace contaminant that can find its way into cannabis products are heavy metals, which are absorbed from the soil into the cannabis plant. Many of these are considered toxic, such as lead, arsenic and mercury. Options for detection include Ultrasonic Nebulizer Inductively Coupled Plasma Optical Emission Spectrometry (USN-ICP-OES) and Mass Spectrometry (ICP-MS) methods, both of which are capable of rapidly analyzing all heavy metals but differ in simplicity and sensitivity.

Moisture content is also another important parameter as excessive moisture can promote mold growth, which can be established using an electronic moisture analyzer like the Shimadzu MOC63u.

Related to this is concern over the presence of mycotoxins, which are toxic metabolites produced by mold. Several of these have been shown to be especially harmful to humans and can be dangerous to immunocompromised patients even at very low concentrations. Therefore, it is critical to have sensitive methods for detecting low levels in cannabis samples. Chromatographic methods available include GC, HPLC and LC-MS/MS.

Chapter 4 – Cannabis Standards

The increasing legal status of cannabis has led in turn to a surge in demand for cannabis testing labs. But cannabis testing is very much an emerging industry and there has been considerable confusion surrounding how such labs should operate. For example, some have been uncertain over whether they were able to provide their services legally due to the continued federal prohibition of the drug. And with no federal oversight into cannabis standards, such as cultivation conditions or acceptable levels of residue solvents and pesticides, states have been left to come up with their own rules, sometimes belatedly following legalization. Labs have also had to contend with uncertainty because of the prospect that current standards become obsolete when state lawmakers decide to change the regulations.

At this year’s Pittcon, the symposium “It’s legal! Now what?” will explore some of these emerging issues. Heather Krug from the Colorado Department of Public Health and Environment will detail the challenges the state has faced since legalizing recreational cannabis. This includes issues such as a lack of scientific evidence over the toxicity of cannabis contaminants, systems for monitoring cannabis lab performance and an absence of industry-accepted standards for labs to work to.

The symposium will also hear from Robert Martin, representing the Association of Commercial Cannabis Laboratories, who will outline their efforts to try to establish evidence-based quality measures for cannabis testing. The talk will touch upon issues faced in pesticides, residual solvents and microbiological testing, as well as consider the future of the cannabis testing industry.

Also at Pittcon 2017, Autumn Karcey from Cultivo, Inc. will discuss the optimal conditions for growing cannabis indoors to facilitate medical research into its effects. This includes consideration of factors such as temperature, humidity and room pressure as well as how to minimize the presence of airborne particulates, pests and pathogens.

Barry Schumbmehl from Fritsch Milling and Sizing, Inc. will also discuss how milling and grinding procedures during sample preparation can help to generate uniform, representative samples suitable for analyzing commercially produced cannabis. He will discuss the variability of analytes between crops and even within the same plant and provide data on how potency can therefore differ between that which is stated and as determined from dried flowers or post-milling.

Chapter 5 – Cannabis Detection and Law Enforcement

As we have seen, analytical techniques have been assisting the regulation of legalized cannabis but the fact still remains that the substance is prohibited in most countries, and is still illegal for recreational use in the majority of US states. Detecting illicit cannabis in biological samples and contraband therefore continues to be a priority and advances in analytical science are helping this to be done with greater accessibility, speed and sensitivity.

One such development is cantilever-enhanced photoacoustic spectroscopy, or CEPAS. At Pittcon 2017, we will hear from Jaakko Lehtinen from Finland-based Gasera Ltd., who were involved in developing this new measurement technique. It can be used in gas phase for detection of volatile organic compounds (VOCs) from drugs or their precursors and in this talk, Lehtinen will discuss how the use of different laser sources can lead to Ppb-level detection of VOCs. He will also outline how photoacoustic spectroscopy is able to distinguish hair samples from cannabis users and non-users, and how THC levels can be determined from cannabis samples using the same set-up.

Pittcon 2017 will be attended by a number of companies whose technologies are providing novel options for drug detection to assist law enforcement.

For example, Advion will be presenting their compact mass spectrometry device. Their team have previously shown how the device, which is intended to make MS more accessible, particularly in labs with limited space, can be used alongside a method called Atmospheric Solids Analysis Probe (ASAP®) combined with atmospheric pressure chemical ionization (APCI) to allow for direct analysis of samples suspected of containing cannabis. The approach was able to produce strong signals for cannabinol, THC/cannabidiolic acid and THC/cannabinol within 20 seconds. Furthermore, they showed that the method could potentially be applied to trace samples from fingertips, as when a person has rolled a cigarette containing cannabis.

Bruker, who will also be presenting at this year’s conference, offer a number of drug testing solutions, including the Toxtyper workflow which facilitates LC-MS drug screening by incorporating a library of over 830 compounds. A team of researchers showed that Toxtyper could be applied to the identification of synthetic cannabinoids. New derivatives of these compounds frequently emerge in slightly modified varieties, sometimes as a deliberate attempt to bypass drug laws, and they can pose a risk to consumer health due to their unknown strength and toxicity. The research team showed using a library of 46 synthetic cannabinoids and nine isotope-labelled analogs, that spiked serum samples could be detected for all substances at concentrations of 0.5 ng/mL or lower.

Bruker also offer an ion mobility spectrometer, called DE-tector, a desktop and portable device, which can detect natural, synthetic, pure and street drugs from samples when present in the low nanogram range.

The company have also shown that it’s Fourier transform-infrared spectrometer, ALPHA, is effective in detecting cutting agents that have been laced into drug samples, and are themselves often illicit and harmful to consumers.

Also at this year’s Pittcon will be Biotage. They have created a range of supported liquid extraction plates and columns called ISOLUTE which can be used in place of traditional liquid-liquid extraction. Not only can the set-up be used to detect cannabinoids in blood and urine samples, but the company have also shown that it can be applied to saliva samples, potentially presenting a quicker and easier way to obtain samples in settings such as traffic accidents or for workplace drug testing.

Thermo Scientific offer a solution for cannabinoid detection in biological samples using their TurboFlow technology – an automated online sample preparation technique – coupled to LC-MS/MS. Their team has shown that the approach is able to quantify THC and its metabolites in spiked blood samples, with a total extraction and analytic runtime of 10.4 minutes. Thermo Scientific say the method, by cutting out sample preparation time, can provide advantages over SPE or liquid-liquid sample preparation.

Conclusion

Against a background of increasingly liberal cannabis laws, regulators and scientists have been trying to keep pace with cannabis as it has gone from a near-universally illicit substance to a burgeoning industry in just a matter of years.

Fortunately, the analytical science industry, drawing on experience in the fields of food, environmental and agricultural science, is rising to the many challenges presented by this revolution.

At Pittcon, taking place in Chicago from 5-9 March, 2017, you can meet the companies at the forefront of innovation in cannabis analysis, regulation and detection. There are also a wealth of talks and symposia during which you can hear directly from the experts who have been involved in developing these technologies and methodologies, and putting them to use in the field.

The many presentations will explore recent developments in cannabis research, law and ethics. The conference will also look to the future, and what it holds for the cannabis testing industry and medicinal uses of the drug. For example, a talk by Kevin Rosenblatt from Integrated Biosource/Cannabis Labs, will consider how medical marijuana fits into another healthcare revolution – personalized medicine. Rosenblatt will look at the potential for genomics, metabolomics and pharmacogenomics to tailor cannabis treatment to patients on an individual basis, and how this could be assisted by analytical techniques like mass spectroscopy.

As this industry tries to make sense of legalized medical and recreational cannabis from an ethical, legal, and analytical perspective, come to Pittcon 2017 to find out where we are now and where we could go next.